MabSelect Xtra * GE Healthcare Life Sciences. Medium characteristics Description. MabSelect family

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1 GE Healthcare Life Sciences Data file AC Affinity chromatography MabSelect Xtra * MabSelect Xtra is a recombinant protein A-based affinity medium engineered to give an exceptionally high dynamic binding capacity for monoclonal antibodies. In addition, MabSelect Xtra is optimized for Fc-fusion proteins. Higher capacity translates directly to lower cost of production. Key performance characteristics of MabSelect Xtra include: High dynamic binding capacity and suitable for highexpression feedstocks Improved process economics through reduced raw materials costs and/or reduced number of cycles Very low, unspecific binding due to high ligand selectivity and matrix hydrophilicity High capacity for many Fc-fusion proteins Medium characteristics Description MabSelect Xtra is a member of the MabSelect* product line of recombinant protein A-based affinity media for capturing monoclonal antibodies. Like its companion product, MabSelect, MabSelect Xtra is based on an innovative, high-flow agarose base matrix, optimized for maximum capacity (Fig ). The base matrix for MabSelect Xtra has increased porosity and decreased particle size relative to MabSelect. The recombinant protein A is coupled to the base matrix at the C-terminal cysteine via a stable thio-ether bond. It is the same ligand and coupling chemistry used in MabSelect. This oriented coupling contributes to the increased capacity seen with these media. Fig 1. MabSelect Xtra captures antibodies from high-expression feedstocks with improved efficiency and economy. MabSelect family The MabSelect family of media for process-scale purification of monoclonal antibodies comprises MabSelect, MabSelect Xtra, MabSelect SuRe*, and MabSelect SuRe LX. MabSelect is designed for high-throughput purification of monoclonal antibodies from large volumes of feed. MabSelect SuRe is composed of the same rigid, high flow agarose matrix, but posesses an alkali-stabilized rprotein A-based ligand. The MabSelect SuRe ligand provides greater stability than conventional rprotein A-based media and allows extended use of.1. M NaOH for cleaning-in-place. MabSelect SuRe LX has been further developed from MabSelect SuRe to give even higher binding capacity at longer residence time. For more information on MabSelect, MabSelect SuRe, and MabSelect SuRe LX, refer to Data files , , and , respectively.

2 Fig. Electron micrographs of a MabSelect Xtra particle. The inset (resolution nm) shows the structure of the high-flow agarose matrix with large pores and high connectivity. MabSelect Xtra is easy to pack and operate in a wide variety of columns from laboratory to production scale. Well-proven packing and cleaning-in-place (CIP) recommendations are available, as are scale-up studies. Table 1 lists the key characteristics of MabSelect Xtra. Table 1. Main characteristics of MabSelect Xtra Composition Highly cross-linked agarose Average particle size 1 d v 7 µm Ligand Recombinant protein A (E. coli) Coupling chemistry Epoxy Dynamic binding capacity Approx. 4 mg human IgG/ml medium (MabSelect approx. 3 mg IgG/mL medium) Chemical stability Stable in all aqueous buffers commonly used in protein A chromatography:.1 M % sodium citrate/hcl (ph 3), 6 M Gua-HCl, 6 M urea, % ethanol, % benzyl alcohol Recommended ph Working range 3 Cleaning-in-Place (CIP) 1 (see Cleaning and sanitization) Recommended mobile phase velocity 3 Temperature stability 4 Delivery conditions Mammalian product Regulatory support 3 cm/h (MabSelect cm/h) C 4 C % ethanol No material of animal origin is contact-free used in the manufacturing process Regulatory Support File available 1 d v is the median particle size of the cumulative volume distribution. Determined at % breakthrough by frontal analysis at a nominal mobile phase velocity of cm/h in a column with a bed height of cm (.4 min residence time). 3 In a packed bed, the maximum recommended operating velocity for MabSelect Xtra is 3 cm/h at cm bed height, with water at C. Operating pressure is less than bar. 4 Recommended long-term storage conditions: C to 8 C, % ethanol. Dynamic binding capacity Lower cost of production is a key processing goal of monoclonal antibody manufacturers. In upstream operations, cell culture expression levels are steadily increasing and have reached multi-gram levels per liter medium in many processes. MabSelect Xtra responds to these improvements in cell culture optimization; the medium is designed for the downstream capture of antibodies from high expression feedstocks. Confirmation of the high dynamic binding capacity (DBC) of MabSelect Xtra compared with competitor high capacity media is seen in Figure 3, where the two media were tested with human IgG. (mg/ml) MabSelect Xtra ProSep**-vA Ultra Residence time (min) Fig 3. At equivalent residence times, MabSelect Xtra has 3% higher dynamic binding capacity than porous, glass-based protein A medium. Feed concentration was 1.1 mg/ml human polyclonal IgG. Data obtained in studies performed at GE Healthcare laboratories The outstanding dynamic binding capacity of MabSelect Xtra is also observed when two different monoclonal antibody feedstocks are tested. At a residence time of.4 min the dynamic binding capacity at % break-through of a humanized IgG 4 (MAb 1) is 4 mg/ml and of a humanized IgG 1 (MAb ) about 3 mg/ml. Increasing the residence time to 4.8 min, increases the dynamic binding capacity by 1% (Fig 4). QB % (mg/ml medium) Polyclonal higg MAb-1 MAb Residence time (min) Fig 4. MabSelect Xtra dynamic binding capacity of immunoglobulins at.4 min residence and 4.8 min, respectively AC

3 Protein A leakage Leakage of protein A from MabSelect Xtra is generally low. However, leakage of protein A is also affected by chromatographic running conditions and the composition of feed stock. A purification study of IgG 4 at pilot scale, which included a blank run and CIP showed leakage levels of. 7. ppm (ng/ mg), see Figure. Results show that the content of host cell protein in the eluates was relatively constant over cycles (Fig 6). However, normalized levels (in ppm) were approximately 6-fold higher for a glass bead-based medium than for MabSelect Xtra. This result suggests a higher degree of nonspecific adsorption to porous glass beads compared to the hydrophilic agarose base matrix in MabSelect Xtra. ProSep va Ultra MabSelect Xtra ppm protein A Protein A HCP conc. (ppm) normalized Cycle no. Run No. 1 Run No. Run No. 3 Fig. The leakage of protein A in the eluted IgG 4 material showed protein A levels between. and 7. ppm (ng protein A/mg IgG). To remove loosely bound protein A, a blank run including CIP is recommended before the first run with antibody feed and after storage of the media. Host cell protein study The level of host cell proteins in the eluate from MabSelect Xtra has been assessed following repeated processing of a Chinese hamster ovary (CHO) supernatant spiked with human IgG. Complete cycles (equilibration, loading, elution, washing, and CIP) were run, after which the level of host cell protein in the eluate was measured using a high sensitivity ELISA protocol. A comparative study was made on a protein A medium attached to a porous glass base matrix. Fig 6. Normalized HCP concentration: a comparison between MabSelect Xtra and a porous glass bead, high-capacity medium. Data obtained in studies performed at GE Healthcare laboratories. Operation Method optimization Method optimization should establish conditions that promote binding the highest amount of target antibody in the shortest time with maximum purity and recovery. In general, all antibodies of human origin, except subclass 3, have a high affinity for protein A. To optimize elution, determine the highest ph that allows efficient desorption of antibody. Step-wise elution is preferred at large-scale as the antibody elutes in a smaller pool volume, thus decreasing buffer consumption and shortening cycle times. PreDictor* 96-well filter plates or miniaturized columns, prefilled with MabSelect Xtra media, are suitable for efficient highthroughput screening of chromatographic conditions during process development. Defined conditions can then be verified and optimized with HiScreen* or HiTrap* prepacked columns AC 3

4 Process development For process development work using MabSelect Xtra in small-scale columns, we recommend HiScreen prepacked columns with a cm bed height. Choose a residence time that meets the demands on capacity and nominal fluid velocity. Make sure the chosen bed height and velocity do not conflict with the large-scale pressure/flow limitations (see Fig 7). Velocity (cm/h) Not recommended area Not recommended area Fig 7. Operating window for MabSelect Xtra: Choosing bed height and operating velocity in terms of residence time, pressure restrictions, and large-scale column packing Bed height (cm) Figure 7 shows the possible operating window with combinations of bed height and operational nominal mobile phase velocity for MabSelect Xtra. The Figure also displays the residence time in minutes for any bed height and velocity. Pressure drop and packing limitations at large scale are also included. The solid line shows the large-scale column pressure restriction (light blue background). The dashed line indicates that operating below cm bed height is not favorable (light blue background). The reason for this is that large diameter columns have a very different aspect ratio, and that packing short wide beds is a greater challenge. Use Figure 7 as a guide when determining suitable bed height and operating velocity in terms of residence time and thus capacity and pressure drop. 1 Cleaning and sanitization MabSelect Xtra is based on the same rprotein A ligand and same coupling chemistry as MabSelect, and similar cleaning-in-place (CIP) protocols can be applied. For MabSelect, a high-throughput screening approach using PreDictor plates has been performed [1]. The most favorable results were obtained using a two-step sequence with reducing agent ( mm 1-Thioglycerol) followed by 1 mm NaOH. Stronger NaOH can be used, but might reduce the lifetime of the media. The two-step CIP protocol was verified in column experiments using CHO cell supernatant with humanized IgG 1. CIP was performed in every cycle using a contact time of 1 min and a blank cycle was run every th cycle for measuring of carryover. 1 CIP cycles were performed with consistent results and no carry-over was observed. Of the one step CIP protocols evaluated in the screening for MabSelect, mm NaOH gave best results but this solution might reduce the lifetime of the media. Addition of salt (e.g., NaCl och Na SO 4 ) to the caustic CIP solution can increase the rprotein A stability but might decrease the cleaning efficiency. Lower NaOH concentrations (-3 mm) was not efficient for cleaning of MabSelect in this specific MAb process. As alternative cleaning protocol using 6 M guanidine hydrochloride can be used, also in combination with reducing agent. To remove hydrophobically bound substances a solution of non-ionic detergent or ethanol is recommended. Recommendations for sanitization of MabSelect Xtra (reducing microbial contamination of the packed bed to a minimum) with a number of well-proven methods are listed in Instruction AC

5 Scale-up After optimizing the antibody purification at laboratoryscale, the process can be scaled up to larger columns. MabSelect Xtra has been successfully packed in largescale columns in our laboratories (Fig 8). As noted in Table 1, at 3 cm or larger diameter scale (where wall effects are no longer a consideration), the maximum recommended operating velocity for MabSelect Xtra is 3 cm/h at cm bed height with a low viscosity buffer. Velocity (cm/h) Pressure (bar) Fig 8. Typical pressure/flow curve obtained in AxiChrom 6, at cm bed height. System/tubing pressure is included in the pressure. Packing methods for large-scale columns Good column packing is essential for any chromatographic process and naturally plays a key role in large-scale commercial manufacture. Proper packing will eliminate concerns such as channeling and compression and ensure a stable bed that performs according to expectations over many processing cycles. At process scale, the preferred packing technique is axial compression. Using AxiChrom* columns, with Intelligent Packing and preprogrammed packing methods for all MabSelect products, is an effective approach. Test results show that MabSelect Xtra is both easy to pack and repack reproducibly at large scale. Table lists recommended large-scale column details. Table. Recommended columns for MabSelect Xtra Column family range Inner diameter (mm) Lab scale: Tricorn*, HiScale* 16, 6, Pilot and production scale: AxiChrom BPG* 3 Chromaflow* 4 8 The pressure rating of BPG 4 is too low to use it with MabSelect Xtra media. Larger pack stations might be required at larger diameters. Methods for large-scale packing of MabSelect Xtra are available from GE Healthcare AC

6 Process economy Lower cost of antibody capture An important, if not the most important, consideration in bioprocessing is the economic aspect of the process. This is an especially important factor considering the anticipated increase in fermentation volumes and expression levels. For a primary recovery step, the purification cost can be broken down into two types of contributions, one related to the number of cycles and the other related to the process time. In the first group, the cost of chemicals including chromatography media, as well as quality control are two important factors that need to be considered. In the second group, labour and facility costs may be predominant factors. The increase in dynamic binding capacity affects process economy because a lower number of cycles leads to a less frequent replacement of protein A media and to a proportionally lower buffer consumption. For example, an increase of 3%, which should be the typical dynamic binding capacity gain of using MabSelect Xtra, results in about % 3% lower overall cost of the capture step (Fig 9). Application purification of IgG 4 The capture of IgG 4 (pi 6. 7.) summarized below was performed on a FineLINE* Pilot 3 column packed with MabSelect Xtra using an ÄKTApilot* chromatography system. The column was packed with MabSelect Xtra to a bed height of cm and run at a nominal fluid velocity of 3 cm/h, which gave a residence time of 4 min. The purification cycle, which included CIP was repeated three times. Figure shows the running conditions and chromatogram of the first run. IgG 4 yield and purity were high over all three runs. Average yield was 94% and the monomer form of the material as determined by analytical gel filtration on Superdex* HR exceeded 99% (Fig 11). The goal of achieving high recovery and product purity with the capture step run at pilot-scale on the FineLINE Pilot 3 column was clearly reached (the eluted antibody contained more than 99% monomer). In addition, the purification step was very consistent; the same separation and analytical results were found in all three runs. This work is further described in Application Note Pilot-scale capture of monoclonal antibody IgG 4 on MabSelect Xtra with high purity and yield. 4 Economic gain (%) % DBC increase 3% DBC increase 4% DBC increase Column: FineLINE Pilot 3 (bed height.1 cm, 1 column volume =193 ml) packed with MabSelect Xtra Sample: IgG 4 clarified NSO supernatant, Sample load:.16 L (. g/l) Buffer A: mm sodium phosphate, 1 mm NaCl, ph 7. Buffer B: mm glycine-hcl, ph 3. Flow rate: 3 cm/h (48 ml/min) Gradient: % % B in CV CIP: mm NaOH, mm Na SO 4, contact time min (46 ml/min) System: ÄKTApilot mau 3 1 Eluted IgG4 ph Contribution of buffer and affinity medium to the overall process cost (%) Fig 9. Effect of an increase in dynamic binding capacity on process economy. The economic gain is shown for a %, 3%, and 4% increase in DBC ml 4. Fig. Capture of IgG 4 on MabSelect Xtra packed in a FineLINE Pilot 3 column. Average yield (94%) and purity (more than 99% monomer) were high over the three runs performed. The NSO cell line feedstock containing the IgG 4 monoclonal antibody was supplied by Lonza Biologics, UK AC

7 Column: Medium: Sample: Tricorn* /3 (bed height 3 cm, 1 column volume = 3.6 ml) Superdex HR IgG 4 purified on MabSelect Xtra from clarified NSO supernatant, Lonza Biologics Sample volume: µl Buffer A: PBS, ph 7.4 Flow rate: 39 cm/h (. ml/min) System: ÄKTAexplorer* mau 6 nm Ordering information Product Pack size Code No. MabSelect Xtra ml ml L L L L HiScreen MabSelect Xtra ml HiTrap MabSelect Xtra 1 ml ml PreDictor MabSelect Xtra 6 μl (4 96-well plates) μl (4 96-well plates) μl (4 96-well plates) ml PreDictor RoboColumn** MabSelect Xtra µl (one row of 8 columns) µl (one row of 8 columns) Fig 11. Dimer/aggregate analyses on Superdex HR of the start material showing clarified IgG 4 supernatant (red), flowthrough (green), and eluate after the capture step (blue). Note that no monomer of IgG 4 was found in the flowthrough (green line). References 1. Grönberg, A. et al., GE Healthcare. Poster at 1st High- Throughput Process Development International Conference, Krakow, Poland (). Related literature Data files MabSelect MabSelect SuRe MabSelect SuRe LX PreDictor 96-well filter plates and Assist software PreDictor RoboColumn HiTrap MabSelect, MabSelect Xtra, MabSelect SuRe HiScreen prepacked columns BPG columns HiScale columns AxiChrom columns Chromaflow columns Application notes MabSelect Xtra IgG 4 purification on ÄKTApilot Handbooks Antibody purification, principles and methods High-throughput process development with PreDictor plates, principles and methods All bulk media products are supplied in suspension in % ethanol. For additional information, please contact your local GE Healthcare representative AC 7

8 For local office contact information, visit GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden imagination at work GE, imagination at work, and GE monogram are trademarks of General Electric Company. * AxiChrom, ÄKTAexplorer, ÄKTApilot, BioProcess, BPG, Chromaflow, FineLINE, HiScale, HiScreen, HiTrap, MabSelect, MabSelect SuRe, MabSelect Xtra, PreDictor, Superdex, and Tricorn are trademarks of GE Healthcare companies. ** All third party trademarks are the property of their respective owners. -11 General Electric Company All rights reserved. First published All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse D Freiburg Germany GE Healthcare Bio-Sciences Corp. 8 Centennial Avenue, P.O. Box 137 Piscataway, NJ USA GE Healthcare Japan Corporation Sanken Bldg., 3--1, Hyakunincho Shinjuku-ku, Tokyo Japan AC 6/ AC